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Impacts of degraded DNA on restriction enzyme associated DNA sequencing ( RADS eq)
Author(s) -
Graham Carly F.,
Glenn Travis C.,
McArthur Andrew G.,
Boreham Douglas R.,
Kieran Troy,
Lance Stacey,
Manzon Richard G.,
Martino Jessica A.,
Pierson Todd,
Rogers Sean M.,
Wilson Joanna Y.,
Somers Christopher M.
Publication year - 2015
Publication title -
molecular ecology resources
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.96
H-Index - 136
eISSN - 1755-0998
pISSN - 1755-098X
DOI - 10.1111/1755-0998.12404
Subject(s) - biology , restriction enzyme , dna , dna sequencing , genomic dna , restriction site , genomic library , genetics , illumina dye sequencing , microbiology and biotechnology , computational biology , base sequence
Abstract Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next‐generation DNA sequencing ( RADS eq, GBS ) is unknown. We experimentally examined the effects of in situ DNA degradation on data generation for a modified double‐digest RADS eq approach (3 RAD ). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish ( Coregonus clupeaformis ) following 0‐, 12‐, 48‐ and 96‐h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12–48 h) performed well. In contrast, the number of RAD tags per individual, number of variable sites, and percentage of identical RAD tags retained were all dramatically reduced when libraries were made using highly degraded DNA (96‐h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2‐ to 16‐fold), and additional read‐length trimming. We conclude that starting DNA quality is an important consideration for RADS eq; however, the approach remains robust until genomic DNA is extensively degraded.